Pullulanase (EC 3.2.1.41) is an enzyme hydrolyzing an α-1,6 linkage of, for example, amylopectin in starch. Pullulanase is an enzyme having a high industrial applicability in the fields of sugar, for example, production of maltooligosaccharides such as glucose, maltose, maltotriose, maltotetraose, maltopentaose and maltohexaose (OLIGOSACCHARIDES, Gordon and Breach Science Publishers, p3), improvement of rice cooking (patent document 1), and the like.
Pullulanase derived from microorganism includes Bacillus sp. APC-9603 (patent document 2), and ones derived from Klebsiella pneumonia (AMANO ENZYME INC.), Bacillus deramificans, Bacillus acidpullulyticus, Bacillus stearothermophilus, Bacillus sectorramus, Bacillus circulans, Bacillus cereus, and Bacillus sectorramus. 
Similar to the other enzymes, when pullulanase is used, concentration of substrate and enzyme, reaction temperatures, reaction time, and the like are adjusted depending upon the applications of use. However, with adjustment of such enzyme reaction conditions alone, it may not be possible to produce intended products or to obtain an expected yield. Thus, it has been necessary to modify the properties themselves of pullulanase.
In order to modify the properties of pullulanase, it is necessary that mutants of pullulanase should be produced, and the activity, substrate specificity, and the like, should be evaluated so as to search for an excellent mutant. However, such processes have required much labor. Patent document 3 discloses one example of a mutant of pullulanase.                Patent document 1: JP H7-289186 A        Patent document 2: JP H5-292962 A        Patent document 3: JP 2002-505108 A        Non-patent document 1: J Mol Biol. 2006 Jun. 9; 359 (3): 690-707        